In Vitro and Xenograft Anti-Tumor Activity of a Halogenated-Xanthene Against Refractory Pediatric Solid Tumors

ABSTRACT

A method of treating a pediatric cancerous solid tumor in a mammalian subject is disclosed that comprises intralesionally administering an amount of a halogenated xanthene or a pharmaceutically acceptable salt thereof, preferably Rose Bengal disodium, that elicits ablation of tumor cells of the administered tumor. Another contemplated method comprises the steps of intralesionally administering an amount of a halogenated xanthene or a pharmaceutically acceptable salt thereof, preferably Rose Bengal disodium, that elicits ablation of tumor cells of the administered tumor and systemically administering a tumor-inhibiting effective amount of a systemic anti-cancer medication that provides synergistic cytotoxicity with the halogenated xanthene. The two administrations can occur concurrently, or one prior to the other.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority from application Ser. No. 62/672,373that was filed on May 16, 2018, whose disclosures are incorporatedherein by reference.

BACKGROUND ART

The types of cancers that develop in children are often different fromthe types that develop in adults. Childhood cancers are often the resultof DNA changes in cells that take place very early in life, sometimeseven before birth. Genetic mutations that initiate cancer developmentcan thus arise during the development of a fetus in utero. Unlike manycancers in adults, childhood cancers are not strongly linked tolifestyle or environmental risk factors.

In addition, children face unique issues during their treatment forcancer, after the completion of treatment, and as survivors of cancer.For example, children may receive more intense treatments, while theircancer and its treatments can have different effects on a child'sgrowing body compared to an adult body. Children can respond differentlyto drugs used to control symptoms in adults.

Adolescents and young adults are often diagnosed with different types ofcancer from either younger children or older adults. The incidence ofspecific cancer types varies widely across the adolescent and youngadult age continuum. Some evidence suggests that adolescents and youngadults with acute lymphoblastic leukemia may have better outcomes ifthey are treated with pediatric treatment regimens than if they receiveadult treatment regimens.

Currently, children with relapsed or metastatic solid tumors such asEwing sarcoma, neuroblastoma, osteosarcoma and rhabdomyosarcoma have alow overall survival rate of less than 30% [1]. Of the pediatric solidtumors, neuroblastoma is the most common extra-cranial cancer inchildren and a leading cause of death in children aged 1-4 years [2].

Neuroblastoma originates from sympathetic nervous tissue and is a veryheterogeneous and complex disease [3]. Recent improvements in treatmentof neuroblastoma have increased 5-year survival rates for non-high riskdisease to over 90% [4]. However, more than 40% of patients presentingwith neuroblastoma are considered high-risk and despite intensivetreatment regimens, 5-year survival rates for these patients are below50% [2, 4]. Additionally, the prognosis for relapsed neuroblastoma isdismal, with a 5-year survival rate of less than 10% [4].

Given the poor survival rates of pediatric patients with relapsed ormetastatic solid tumors such as, in particular those with high-risk andrelapsed neuroblastoma, novel therapeutic approaches for the treatmentof these malignancies are urgently needed.

One useful anti-cancer agent group for adult cancerous tumors are thehalogenated xanthenes, or the pharmaceutically acceptable salts thereof.See, U.S. Pat. Nos. 6,331,286, 7,390,668, and 7,648,695. Of thosehalogenated xanthenes, Rose Bengal disodium,(4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein disodium; RB) hasbeen found to be particularly effective and easily utilized. Because ofthe often times very different behavior of adult tumors from pediatrictumors, it is not known whether RB and similar halogenated xantheneswould be similarly effective when used against pediatric canceroustumors.

PV-10 is a sterile 10% solution of (RB), in 0.9% saline, that has beenused clinically to measure liver function in infants [5]. Previousstudies have shown that PV-10 accumulates in lysosomes [6] and inducescell death in a range of adult cancers [7-11].

In a phase II clinical trial for patients with refractory metastaticmelanoma, intralesional (IL) injection of PV-10 induced tumor regressionwith an overall response rate of 51% [12]. PV-10 also demonstratedefficacy in combination with radiotherapy in a phase II clinical trialfor patients with in-transit or metastatic melanoma, with an overallresponse rate of 86.6% [13]. In addition to inducing direct cancer celldeath, PV-10 has also been shown to induce a tumor-specific immuneresponse in both mouse studies [7, 8, 14] and clinical trials [10, 12,14, 15, 16].

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates a method of treating a pediatriccancerous solid tumor in a mammalian subject. One method contemplatesintralesionally administering an amount of a halogenated xanthene or apharmaceutically acceptable salt thereof that elicits ablation of tumorcells of the administered tumor. A second method comprises the steps ofintralesionally administering an amount of a halogenated xanthene or apharmaceutically acceptable salt thereof that elicits ablation of tumorcells of the administered tumor. A tumor-inhibiting effective amount ofa systemic anti-cancer medication that provides synergistic cytotoxicitywith the halogenated xanthene is also administered systemically to themammalian subject.

The two medications can be administered concurrently, or one can beadministered before the other by about one to about four weeks. It ispreferred to carry out the intralesional administration prior to thesystemic administration by about one to about four weeks.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure

FIG. 1, including FIGS. 1A-1E, illustrates that PV-10 decreases cellviability in pediatric solid tumor cell lines. Different pediatric solidtumor cell lines (Ewing sarcoma, neuroblastoma, osteosarcoma andrhabdomyosarcoma), normal fibroblast cell lines and a primary bonemarrow sample were treated with increasing concentrations (3.125-400 μM)of PV-10 for 96 hours. Cell viability was measured by alamar blue®assay. Percent cell viability was normalized to corresponding treatmentwith PBS (vehicle control). Mean percentages of cell viability werecalculated from three separate studies and standard errors of the meansare shown.

FIG. 2, including FIGS. 2A and 2B, are photomicrographs showing thatPV-10 is cytotoxic to neuroblastoma cell lines. In FIG. 2(A),neuroblastoma cell lines SK-N-AS, SK-N-BE(2) and IMR5, and the closelyrelated neuroepithelioma cell line SK-N-MC were treated with either PBS(vehicle control), 50 μM or 100 μM PV-10 for 96 hours and observed byphase-contrast light microscopy. Studies were performed three times andrepresentative images are shown. Scale bar equals 100 μm. In FIG. 2(B),neuroblastoma cell lines SK-N-AS, SK-N-BE(2) and IMR5, and theneuroepithelioma cell line SK-N-MC were treated with either PBS (vehiclecontrol) or 100 μM PV-10 and observed by time-lapse video microscopy.Images were captured every 30 minutes for 48 hours. Cell number wascounted and normalized to cell number at 0 hours. At least 350 cellswere counted per treatment per study. Mean percentages of cell numberscalculated from three separate studies and standard errors of the meansare shown.

FIG. 3 illustrates that PV-10 disrupts lysosomes in cancerous cells.Neuroblastoma cell lines SK-N-AS, SK-N-BE(2) and IMR5 were treated witheither PBS (vehicle control) or 100 μM PV-10 16 hours. Live cells werestained with nucleic acid stain Hoechst 33342 and LysoTracker® GreenDND-26, which concentrates and fluoresces in acidic organelles, andobserved by fluorescence microscopy. Scale bar equals 20 μm. Datapresented are representative of three separate studies.

FIG. 4, including FIGS. 4A-4E, shows that PV-10 increases the percentageof cells in G1 phase of the cell cycle and induces cell death byapoptosis. In FIGS. 4A-4D, neuroblastoma cell lines SK-N-AS and IMR5were treated with either PBS (vehicle control), 50 μM or 100 μM PV-10for either 16 or 24 hours, stained with DAPI and analyzed by flowcytometry to detect cell cycle phase. Mean percentages of cells ineither G1, S or G2/M phases of the cell cycle were calculated from threeseparate studies and standard errors of the means are shown. In FIG. 4E,neuroblastoma cell lines SK-N-AS, SK-N-BE(2) and IMR5, and theneuroepithelioma cell line SK-N-MC were treated with either PBS (vehiclecontrol), 75 μM or 100 μM PV-10 for 24 hours. Total cell lysates wereprepared and analyzed by western blotting to detect levels of total andcleaved poly-ADP ribose polymerase (PARP), caspase 3, caspase 7 andcaspase 9. Actin was used as a loading control. Molecular masses areindicated in kilodaltons (kDa). Data presented are representative of twoseparate studies.

FIGS. 5A-5E illustrate that PV-10 treatment is synergistic withdifferent anti-cancer agent treatments. Neuroblastoma cell linesSK-N-AS, SK-N-BE(2) and IMR5, neuroepithelioma cell line SK-N-MC and thenormal fibroblast cell line BJ (FIGS. 5A-5E, respectively) were treatedwith 0.1 μM of seven different anti-cancer agents either alone or incombination with 50 μM PV-10. Cells were treated for 96 hours and cellviability was measured by alamar blue®. Percent cell viability wasnormalized to treatment with PBS (vehicle control). Mean percentages ofcell viability calculated from two separate studies and standard errorsof the means are shown.

FIG. 6, including FIGS. 6A and 6B, shows that PV-10 treatment enhancesthe effect of irradiation. Neuroblastoma cell lines SK-N-AS (FIG. 6A)and IMR5 (FIG. 6B) were pre-treated with either PBS (vehicle control) or50 μM PV-10 for 4 hours. Cells were then irradiated with either 0.5, 1or 2 Gy and cultured for a further 92 hours. Cell viability was measuredby alamar blue®. Mean percentages of cell viability calculated fromthree separate studies and standard errors of the means are shown.Asterisks show significant differences, paired student's t-test, p<0.05.

FIG. 7, including FIGS. 7A-7F, illustrates that PV-10 treatment inducestumor regression in vivo. CB17 SCID mice (n=4 per group) weresubcutaneously injected on the right flank with either IMR5-mCherryFlucor SK-N-AS-mCherryFluc cells. When tumor size was at least 5×5 mm,tumors were injected with either 50 μl PBS (vehicle control), 25 μlPV-10 or 50 μl PV-10. In FIG. 7A, IMR5-mCherryFluc tumor growth wasmeasured using a Vernier caliper. Arrow indicates treatment day (day 6).Mean tumor size and standard error of the mean are shown. Asterisks showsignificant differences, unpaired student's t-test, p<0.05. In FIG. 7B,IMR5-mCherryFluc tumor growth was measured using the Xenogen IVIS® 200system to detect bioluminescent signal following intraperitonealinjection with D-luciferin. Mean tumor size and standard error of themean are shown. FIG. 7C shows a survival curve for mice withIMR5-mCherryFluc tumors. Asterisks show significant differences,Log-rank (Mantel-Cox) test, p<0.05. In FIG. 7D, SK-N-AS-mCherryFluctumor growth was measured over time in days using a Vernier caliper.Arrow indicates treatment day (day 14). Mean tumor size and standarderror of the mean are shown. In FIG. 7E, SK-N-AS-mCherryFluc tumorgrowth at zero, 12 and 15 days post treatment was measured using theXenogen IVIS® 200 system to detect bioluminescent signal followingintraperitoneal injection with D-luciferin. Mean tumor size and standarderror of the mean are shown. FIG. 7F shows a survival curve for micewith SK-N-AS-mCherryFluc tumors.

FIG. 8 shows a group of mouse photographs in each experimental groupdescribed by FIG. 7 where the bioluminescent image depicts active flanktumors in each of the experimental groups (PBS Control, PV-10 25 μL andPV-10 50 μL dose) after day 6, day 12 and day 17.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention contemplates a method of treatment of a pediatricsolid tumor with a pharmaceutical composition that contains atumor-inhibiting effective amount of a halogenated xanthene orpharmaceutically acceptable salt thereof such as Rose Bengal (RB,4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein disodium) into thetumor (intralesionally). The Rose Bengal-containing composition can beutilized as the sole treating agent, but in some preferred embodiments,RB is preferably coupled with another anti-tumor modality such as asystemic anti-cancer medication that can be a small molecule(non-proteinaceous, less than about 1000 grams/mole) or a proteinaceousmolecule such as an antibody or an enzyme, ionizing radiation therapy orso-called checkpoint inhibition antibody therapy. As shown herein,several of these combinations have exhibited synergistic toxicitiestoward pediatric tumors.

Halogenated Xanthene

A contemplated halogenated xanthene such as of Rose Bengal(4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein) that isparticularly preferred, or another halogenated xanthene, includingerythrosin B, phloxine B,4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein,2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein,4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein,2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein,4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein,2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein,4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein,4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein,4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein is present dissolved ordispersed in an appropriate pharmaceutical composition.

A preferred form, Rose Bengal disodium, has the following structuralformula:

Certain details of this preferred embodiment for a contemplatedcomposition are described in U.S. Pat. Nos. 5,998,597, 6,331,286,6,493,570, and 8,974,363, whose disclosures are incorporated byreference herein in their entireties.

Delivery of the halogenated xanthene component of a contemplatedcomposition is most favorable when the composition has a pH value closeto physiologic pH (i.e., approximately pH 7), and especially when the pHvalue is greater than about 4, thereby assuring that a halogenatedxanthene remains in dibasic form in the composition. Thus, in apreferred embodiment, the pH value of the composition is about 4 toabout 10, and more preferably about 5 to about 9, and most preferablyabout pH 6 to about pH 8.

A hydrophilic vehicle is preferred for the medicament to maximizepreference for partitioning of the halogenated xanthene component intotissue. Accordingly, in a preferred embodiment, the vehicle contains aminimum of non-hydrophilic components that might interfere with suchpartitioning.

Accordingly, a preferred formulation of the composition contains, in ahydrophilic, preferably water-containing, vehicle a halogenated xanthenesuch as of Rose bengal(4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein) that isparticularly preferred, or another halogenated xanthene, includingerythrosin B, phloxine B,4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein,2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein,4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein,2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein,4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein,2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein,4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein,4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein,4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein in an appropriatepharmaceutical composition.

A preferred form, Rose Bengal disodium, has the following formula:

Certain details of this preferred embodiment for the pharmaceuticalcomposition are described in U.S. Pat. Nos. 5,998,597, 6,331,286,6,493,570, and 8,974,363, whose disclosures are incorporated byreference herein in their entireties.

A contemplated halogenated xanthene-containing composition typicallycontains the halogenated xanthene at a concentration of about 0.1% (w/v)to about 20% (w/v) in an aqueous medium. The pharmaceutical halogenatedxanthene-containing composition preferably includes a water-solubleelectrolyte comprising at least one cation selected from the groupconsisting of sodium, potassium, calcium and magnesium and at least oneanion selected from the group consisting of chloride, phosphate andnitrate, wherein the electrolyte is at a concentration of between about0.1% (w/v) and about 2% (w/v).

A third ingredient is a water-soluble electrolyte selected from sodium,potassium, calcium and magnesium chlorides, phosphates, and nitrates,wherein the electrolyte is present at a concentration of about 0.1 toabout 2% by weight, or alternately at a level sufficient to provide anosmolality of greater than approximately 100 mOsm/kg up to about 600mOsm/kg. More preferably, the osmolality of the medicament compositionis greater than 250 mOsm/kg, and most preferably approximately 300-500mOsm/kg. The electrolyte is preferably sodium chloride. The electrolyteis preferably present at a concentration of about 0.5 to about 1.5%, andeven more preferably at a concentration of about 0.8 to about 1.2%, andmost preferably at a concentration of approximately 0.9% as is presentin physiological saline.

The aqueous medium of the composition is preferably only water thatmeets the criteria for use in injection. Up to about 20 percent byvolume of the vehicle can be one or more C₁-C₆ mono- or polyhydricalcohols such as methanol, ethanol, propanol, isopropanol, butanol,sec-butanol, glycerol, ethylene glycol, propylene glycol,1,2-butanediol, 2,3-butanediol, erytritol, threitol, trimethylolpropane,sorbitol and the like. More preferably, an alcohol is present in acontemplated composition at less than about 10 percent by volume of thevehicle, and more preferably at less than about 5 percent by volume.

Looked at alternatively, the present invention utilizes a compound ofFormula 1, below, in which R₁ is independently F, Cl, Br, I, H or C₁-C₄alkyl; R₂, R₃, R₄, and R₅ are independently Cl, H or I with at least onesubstituent selected from R₂, R₃, R₄, R₅ being I and at least one is Clor H; and R₆ is independently H or C₁-C₄ alkyl; R¹¹ is H or C₁-C₄ alkyl;R¹² is H or C₁-C₇ acyl; and all (a) tautomeric forms; (b) atropisomers,(c) closed lactone forms as depicted in Formula 2 (below), (d)enantiomers of lactone forms depicted in Formula 2, and (e)pharmaceutically acceptable salts thereof.

The term “physiologically acceptable salt” “pharmaceutically acceptablesalt” in their various grammatical forms refer to any non-toxic cationsuch as an alkali metal, alkaline earth metal, and ammonium saltcommonly used in the pharmaceutical industry, including the sodium,potassium, lithium, calcium, magnesium, barium, ammonium and protaminezinc salts, which can be prepared by methods known in the art. Acontemplated cation provides a water-soluble xanthene salt. Preferably,the salts are sodium, potassium, calcium and ammonium in either the monoor dibasic salt form. The reader is directed to Berge, J. Pharm. Sci.1977 68(1):1-19 for lists of commonly used physiologically (orpharmaceutically) acceptable acids and bases that form physiologicallyacceptable salts with pharmaceutical compounds.

The pH value of the halogenated xanthene pharmaceutical composition canbe regulated or adjusted by any suitable means known to those of skillin the art. The composition can be buffered or the pH value adjusted byaddition of acid or base or the like. As the halogenated xanthenes, orphysiologically acceptable salts thereof, are weak acids, depending uponhalogenated xanthene concentration and/or electrolyte concentration, thepH value of the composition may not require the use of a buffer and/orpH value-modifying agent. It is especially preferred, however, that thecomposition be free of buffer, allowing it to conform to the biologicalenvironment once administered.

It is also preferred that the pharmaceutical composition not include anypreservatives, many of which can deleteriously interfere with thepharmaceutical composition or formulation thereof, or may complex orotherwise interact with or interfere with the delivery of thehalogenated xanthene composition active component. To the extent that apreservative is used, imidurea is a preferred preservative as it doesnot interact with halogenated xanthenes, either in the pharmaceuticalcomposition or upon administration.

A contemplated treatment method is utilized on a mammal in need thereof.A treated mammal can be a primate such as a human, an ape such as achimpanzee or gorilla, a monkey such as a cynomolgus monkey or amacaque, a laboratory animal such as a rat, mouse or rabbit, a companionanimal such as a dog, cat, horse, or a food animal such as a cow orsteer, sheep, lamb, pig, goat, llama or the like.

Each contemplated composition is typically administered repeatedly invivo to a mammal in need thereof until the treated solid cancerous tumoris diminished to a desired extent, such as cannot be detected. Thus, theadministration to a mammal in need can occur a plurality of times withinone day, daily, weekly, monthly or over a period of several months toseveral years as directed by the treating physician.

A contemplated halogenated xanthene compound when injected directly intoa tumor is typically taken up by and accumulates in the cancer cells'lysosomes and induces cell death by apoptosis or another mechanism. Incausing cancer cell death, the cells disintegrate or ablate. It isbelieved that that ablation of cell fragments specifically stimulatesthe mammal's immune system to the antigens displayed on the ablated cellfragments such that tumors distant from the site of intratumoral(intralesional) injection are recognized and are also killed.

As has also been noted previously, a preferred contemplated compositionof a halogenated xanthene or a pharmaceutically acceptable salt thereofis referred to as PV-10™. PV-10™ is a sterile 10% solution of RoseBengal (RB, 4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluoresceindisodium) in 0.9% saline.

A tumor-inhibiting effective amount of a systemic anti-cancer medicationthat provides synergistic cytotoxicity when used in combination with thehalogenated xanthene is administered to the mammalian subject in needand can be formulated using usual liquid, gel, solid or other formats.Thus, it is to be understood that a systemic anti-cancer medication canillustratively be administered orally as by tablet or liquidcomposition, by injection i.v., i.m., s.c., intraperitoneally, viaionizing radiation, or by any other form that provides an effectiveamount of the anti-cancer medication to the subject.

Systemic Anti-Cancer Medication

Systemic anti-cancer medication that is a small molecule(non-proteinaceous, less than about 1000 grams/mole) or a largerproteinaceous molecule, is administered to the subject mammal to betreated such that the medication spreads throughout the subject's bodyas compared to the localized administration that occurs with anintralesional administration of a halogenated xanthene. Illustrativesmall molecule anti-cancer medications include doxorubicin, etoposide,vincristine, cisplatin, irinotecan and cytarabine that were used herein,whereas an exemplary proteinaceous molecule is asparaginase. Of thosemedications, doxorubicin, etoposide and vincristine appeared tosynergize with treatment with a sub-lethal dose of PV-10, and arepreferred.

A useful effective dosage of a small molecule, systemic anti-cancermedication is the dosage set out in the labeling information of a FDA-,national- or international agency-approved medication. Typically,monotherapy dose schedules are set by determining the maximum tolerateddose (MTD) in early-stage clinical trials. The MTD (or a close variationthereon) is then promulgated to later-stage clinical trials forassessment efficacy and more detailed assessment of safety. These MTDsfrequently become the established therapeutic dose upon completion ofclinical testing. However, because the small molecule, systemicanti-cancer medication is contemplated for use with PV-10, a MTD is themaximal amount that would be used, and that amount is to be titrateddownward following usual procedures.

Exemplary dosing schedules for a number of systemic anti-cancermedications that can be combined in the present invention with localizedPV-10 therapy are provided in Table A, below. It is noted that severalof the medications listed below are “small molecules” as defined above,whereas others are large, proteinaceous molecules such as antibodies.They are nonetheless administered systemically.

The proteinaceous anti-cancer medications noted below typically inhibitan inflammatory response caused by chemokines such as the TNF family andthe interleukin family.

TABLE A Exemplary systemic immunomodulatory or targeted anticanceragents Systemic Agent Typical Dose Schedule adalimumab 80 mg initialdose followed in 1 week by 40 mg every other week SQ brodalumab 210 mgsubcutaneously (SC) at Weeks 0, 1, and 2, then 210 mg SC q2wkcertolizumab pegol 400 mg initially and at weeks 2 and 4 followed by 200mg every other week or 400 mg Q4 weeks maintenance SQ etanercept 50 mgtwice weekly for 3 months followed by 50 mg once weekly SQ golimumab 50mg once a month SQ guselkumab 100 mg subcutaneous injection once every 8weeks, after starter doses at weeks 0 and 4 infliximab 5 mg/kg given asan IV induction regimen at 0, 2, and 6 weeks followed by a maintenanceregimen of 5 mg/kg every 8 weeks thereafter ixekizumab 160 mg initialdose followed Q2 weeks with 80 mg until week 12 then 80 mg Q4 weeks SQsarilumab 200 mg every 2 weeks as a subcutaneous injection secukinumab300 mg every week for 4 weeks then 300 mg every 4 weeks SQ ustekinumabLess than 100 kg: 45 mg initially, week 4 followed by 45 mg every 12weeks SQ More than 100 kg: 90 mg initially, week 4 followed by 90 mgevery 12 weeks SQ apremilast Titrated dose over 5 days to work up to 30mg twice daily PO methotrexate Weekly single oral, IM or IV 10 to 25 mgper week or divided 2.5 mg dose at 12 hour intervals for three dosescyclosporine Initial dose 2.5 mg/kg/day taken twice daily as divided(BID); dose titrated up to 4 mg/kg/day BID if response and laboratoryabnormalities don't ensue. azathioprine Used off label for skindiseases, 1.0 mg/kg oral or IV as a single dose or twice a day, dosemaximum is 2.5 mg/kg/day.

Because of additive or synergistic effects, the combination therapiesand method of treatment of the present invention generally permit use ofthe systemic agent at a level at or below the typical dose schedule forthe systemic agent, such as those described in Table A, when used with alocal topical therapy, such as that described below. However, the dosingschedules provided in Table A provide a useful guide for beginningtreatment from which dosages can be titrated to lessened amounts as seenappropriate by the physician caring for a given patient.

Ionizing Radiation Treatment

The results reported herein show that combining treatment of PV-10 withionizing radiation also enhanced the cytotoxicity of the treatment as awhole. For the in vitro studies here, the neuroblastoma cells were firstcontacted with a sub-lethal dose of PV-10 for a time period of fourhours and then irradiated with dosages of 0.5, 1 or 2 Gray of ionizingradiation.

It is to be understood that this treatment regimen was illustrative andhas proven the concept of this combination of treatments. Workers ofordinary are able to utilize these effects and scale the treatmentaccordingly.

Checkpoint Antibody Inhibitors

A still further combination treatment regimen utilizes theadministration of PV-10 and a checkpoint antibody inhibitor, that can beviewed as a special systemic anti-cancer medication. A useful checkpointantibody inhibitor is a humanized monoclonal antibody whoseadministration permits the immune system to recognize the cancer cellsas foreign and assist in eliminating those cancer cells from the body.

Some checkpoint inhibitor antibodies target the PD-1 (programmed celldeath protein 1) receptor on the surface of T cells or the ligand forthat receptor PD-L1. Exemplary of these monoclonals are pembrolizumaband nivolumab that inhibit PD-1. Two antibodies that target PD-L1 areatezolizumab, avelumab and durvalumab. Another group of checkpointinhibitor monoclonal antibodies includes ipilimumab tremelimumab thattarget CTLA-4, a protein receptor that downregulates the immune system.

These anti-cancer medications are also usually used systemically. TheMTD as described in the package labels for these medications can againbe a starting dosage that is typically titrated downward during trialsas discussed previously.

Dosing

A PV-10 companion systemic medication can be administered as often as isneeded or tolerated by the recipient subject. Small molecule medicationstypically have relatively short in vivo half-lives or minutes to days.On the other hand, the checkpoint inhibitor antibodies often have invivo half-lives of one to three weeks.

The in vivo biological lifetime of Rose Bengal is understood to be a fewminutes in the rat. However, because intralesional PV-10 administrationis known to induce a T cell immune enhancement, the effect of anadministration of PV-10 can last for months or more, via memory T cells.

As a consequence of the various half-lives of the compositional regents,it is preferred to treat first with PV-10 and then one or morecombination medicaments. The combination medicament is preferablyadministered about 1 to about 4 weeks after the administration of thePV-10 so that the induced immune activation can at least begin.

Pretreatment with a systemic anti-cancer medication can also be useful.Here the systemic anti-cancer agent can initiate the immune responsethat is enhanced by later treatment with PV-10. Here, it is alsopreferred that a time between the first two treatment be about 1 toabout 4 weeks.

The two medications can also be administered at about the same time,concurrently, which can be simultaneously to within about one week ofeach other.

Results

PV-10 Inhibits Growth of Pediatric Solid Tumor Cell Lines

Ewing sarcoma, neuroblastoma, osteosarcoma, rhabdomyosarcoma and normalfibroblast cell lines and a normal primary bone marrow sample weretreated with different concentrations of PV-10 (3.125-400 μM) for 96hours and cell viability was measured using alamar blue® (FIGS. 1A-1E)to determine the effects of PV-10 on pediatric solid tumors. PV-10decreased cell viability in a concentration-dependent manner in all celllines tested. IC₅₀ values were calculated for all solid tumor cell linesexamined. Table 1, below, shows values for PV-10 treated pediatric solidtumor cell lines 96 hours post-treatment. As is seen, the values rangedfrom 45-108 μM, with a mean of 70 μM.

TABLE 1 Cell Line Cell Type PV-10 IC₅₀ μM SK-PN-DW Ewing sarcoma 80SK-ES Ewing sarcoma 45 SK-N-AS Neuroblastoma 85 LAN1 Neuroblastoma 80SK-N-BE(2) Neuroblastoma 73 IMR5 Neuroblastoma 73 SHEP Neuroblastoma 73SK-N-SH Neuroblastoma 65 SK-N-MC Neuroepithelioma 45 143B Osteosarcoma108 HOS Osteosarcoma 75 RD Rhabdomyosarcoma 56 RH30 Rhabdomyosarcoma 51

By contrast, IC₅₀ values for the normal fibroblast cell lines andprimary bone marrow samples were higher and ranged from 73-143 μM, witha mean of 104 μM (Table 2), below. Table 2 provides one-half maximalinhibitory concentration (IC₅₀) values for PV-10 treated normalfibroblast cell lines and a primary bone marrow sample 96 hourspost-treatment.

TABLE 2 PV-10 IC₅₀ Cell Line Cell Type (μM) BJ Normal fibroblast(foreskin) 143 Primary bone Normal primary fibroblasts 136 marrow WI38Normal fibroblast (lung) 93 WI38 hTERT Normal fibroblast (lung) 75 hTERTtransformed BJ hTERT Normal fibroblast (foreskin) 73 hTERT transformed

PV-10 is Cytotoxic to Neuroblastoma Cell Lines

Having identified that PV-10 is cytotoxic to pediatric solid tumor celllines, neuroblastoma was focused upon because it is the most commonextra-cranial cancer in children. Whether PV-10 was cytotoxic orcytostatic to neuroblastoma cell lines was investigated. Four differentcell lines were chosen for study, three neuroblastoma cell lines[SK-N-AS, SK-N-BE(2) and IMPS] that have different mutations anddifferent sensitivities to PV-10 based on IC₅₀ values, and oneneuroepithelioma cell line (SK-N-MC) that was very sensitive to PV-10based on its IC₅₀ value.

Cells were treated with either PBS (vehicle control), 50 μM or 100 μMPV-10 for 96 hours and observed by phase-contrast light microscopy (FIG.2A). Cells treated with PBS grew to confluency. Cells treated with 50 μMPV-10 did not grow to confluency but few dead cells were observed.

By contrast, few cells remained attached to the plate followingtreatment with 100 μM PV-10, indicating that 100 μM PV-10 was cytotoxicto all cell lines. However, the different cell lines appeared to havedifferent sensitivities to PV-10, with SK-N-AS cells being mostresistant to treatment and SK-N-MC being most sensitive to treatment.

Neuroblastoma Cell Lines Display Different Sensitivities to PV-10

The different sensitivities of the four cell lines (SK-N-AS, SK-N-BE(2),IMR5 and SK-N-MC) to PV-10 were investigated using time-lapse videomicroscopy to quantify the percentage of cells attached to the platefollowing 12, 24, 36 and 48 hours of treatment with 100 μM PV-10. Thenumber of cells attached post-treatment was normalized to cell number atzero hours (FIG. 2B).

SK-N-AS cells were most resistant to treatment, at 12 hours 89% and at48 hours 41% of cells were attached. SK-N-MC cells were most sensitiveto treatment, at 12 hours 3.5% and at 48 hours 0% of cells wereattached. IMR5 cells were more sensitive to treatment at 12 and 24 hours(16 and 7% of cells attached respectively) by comparison to SK-N-BE(2)cells (54 and 14% of cells attached respectively), but by 36 hours asimilar percentage of cells were attached for both cell lines (3% ofSK-N-BE(2) and 2% of IMR5 cells).

These data showed that at early times post-treatment, SK-N-MC cells weremost sensitive to treatment, IMR5 were more sensitive to treatment thanSK-N-BE(2) cells and that SK-N-AS cells were most resistant totreatment.

Treatment with PV-10 Disrupts Lysosomes

Previously, PV-10 had been shown to induce loss of lysosome integrity[6]. SK-N-AS, SK-N-BE(2) and IMR5 cells were therefore treated witheither PBS (vehicle control) or 100 μM PV-10 for 16 hours. Live cellswere stained with the nucleic acid stain Hoechst 33342 and LysoTracker®Green DND-26 that concentrates and fluoresces in acidic organelles, andthe cells were observed by fluorescence microscopy (FIG. 3).

In PBS-treated cells and SK-N-AS PV-10-treated cells, lysosomes werevisible as specific foci. However, in PV-10 treated SK-N-BE(2) and IMR5cells, those foci were no longer visible.

PV-10 Treatment Increases the Percentage of IMR5 Cells in G1 Phase ofthe Cell Cycle

The effect of PV-10 upon the cell cycle by flow cytometry was analyzed(FIGS. 4A-4D) to further determine target modulation of PV-10. The mostresistant (SK-N-AS) and most sensitive (IMR5) neuroblastoma cell lineswere treated with either PBS (vehicle control), 50 or 100 μM PV-10 foreither 16 or 24 hours.

PV-10 had no effect on the cell cycle of SK-N-AS cells. By contrast, 100μM PV-10 increased the percentage of IMR5 cells in G1 phase. At 16hours, the percentage of IMR5 cells in G1 phase post-treatment with 100μM PV-10 increased by 28% when compared to cells treated with PBS.Similarly, at 24 hours, there was a 30% increase in cells in G1 phasepost-treatment with 100 μM PV-10, by comparison to un-treated cells.

Treatment with PV-10 Induces Apoptosis

Western blot analysis was then performed to investigate if PV-10 treatedcells were undergoing apoptosis. SK-N-AS, SK-N-BE(2), IMR5 and SK-N-MCcells were treated with either PBS (vehicle control), 75 or 100 μM PV-10for 24 hours. Total cell extracts were analyzed by western blotting todetect levels of total and cleaved poly-ADP ribose (PARP), total andcleaved caspase 3, total and cleaved caspase 7, and actin (loadingcontrol) (FIG. 4E).

PV-10 treatment showed a concentration-dependent cleavage of PARP.Treatment with 100 μM PV-10 induced PARP cleavage in all cell lines,with lower total protein and total PARP levels in SK-N-MC cells (thecell line most sensitive to PV-10). SK-N-AS and SK-N-BE(2) cells treatedwith 75 μM PV-10 showed less PARP cleavage than cells treated with 100μM PV-10, whereas IMR5 cells had similar levels of PARP cleavage whentreated with 75 and 100 μM PV-10. More total PARP was present in SK-N-MCcells treated with 75 μM PV-10 by comparison to cells treated with 100μM.

Activation of caspases 3, 7 and 9 was dependent on PV-10 concentrationand cell line. Cleaved caspase 3 was present in IMR5 cells treated with100 μM PV-10. Levels of total caspase 7 were lower in SK-N-BE(2) cells.

IMR5 and SK-N-MC cells were treated with both 75 and 100 μM PV-10 andcleaved caspase 7 was detected in 100 μM PV-10 treated SK-N-AS andSK-N-BE(2) cells and 75 and 100 μM PV-10 treated IMR5 cells. Levels oftotal caspase 9 were lower in 75 and 100 μM PV-10 treated SK-N-BE(2)cells and cleaved caspase 9 was detected in 75 and 100 μM PV-10 treatedIMR5 cells. These data indicated that PV-10 was inducing cell death byapoptosis.

PV-10 is Synergistic with Different Anti-Cancer Agents

To determine which commonly used small molecule systemic anti-cancermedications could be combined with PV-10 to enhance cytotoxicity,neuroblastoma cell lines SK-N-AS, SK-N-BE(2) and IMR5, neuroepitheliomacell line SK-N-MC and the normal fibroblast cell line BJ were firstscreened against a panel of seven conventional chemotherapy agents,having different mechanisms of action (FIGS. 5A-5E). All agents werescreened at 0.1 μM, alone and in combination with a sub-cytotoxicconcentration of 50 μM PV-10. Cell viability was determined using alamarblue® assay, 96 hours post-treatment.

Based on these results, the agents that showed the largest increase incytotoxicity when combined, and which had a smaller effect on BJ cells,were selected for further study to determine combination indices (CI)and synergy [18]. Agents evaluated for CI studies in SK-N-AS,SK-N-BE(2), IMR5 and SK-N-MC cells were doxorubicin, etoposide andvincristine.

Table 3, below, provides combination indices for neuroblastoma celllines (SK-N-AS, SK-N-BE(2) and IMR5) and the neuroepithelioma cell lineSK-N-MC treated with either doxorubicin, etoposide or vincristine eitheralone or in combination with 50 μM PV-10 for 96 hours.

TABLE 3 Cell Line Doxorubicin Etoposide Vincristine SK-N-AS 0.77 0.170.35 SK-N-BE(2) 0.72 0.66 0.1 IMR5 0.38 0.65 0.2 SK-N-MC 0.42 0.58 0.43

All agents demonstrated synergism with 50 μM PV-10 in each of the celllines assayed. As is seen, CI values ranged from 0.42-0.77 fordoxorubicin, 0.17-0.66 for etoposide and 0.1-0.43 for vincristine.

PV-10 Induces Radiosensitivity in Neuroblastoma Cell Lines

In addition to commonly used chemotherapies, whether PV-10 enhanced theeffect of treatment with ionizing radiation (IR) in SK-N-AS (FIG. 6A)and IMR5 (FIG. 6B) cells was investigated. Cells were pre-treated for 4hours with either PBS (vehicle control) or 50 μM PV-10 and thenirradiated with either 0.5, 1 or 2 Gray (Gy). Cell viability wasmeasured by alamar blue® 96 hours after initial treatment.

Pre-treatment with 50 μM PV-10 enhanced the effect of IR in both SK-N-ASand IMR5 cells. For SK-N-AS cells, cell viability decreased by 54.8%,58.7% and 60% when cells were pre-treated with PV-10 for 4 hours priorto irradiation with either 0.5 Gy, 1 Gy or 2 Gy, respectively. For IMR5cells, cell viability decreased by 24%, 21% and 13% when cells werepre-treated with PV-10 for 4 hours prior to irradiation with either 0.5Gy, 1 Gy or 2 Gy, respectively.

PV-10 Induces Tumor Regression In Vivo

To determine if PV-10 is also active in vivo, we characterised theeffect of PV-10 intratumoral injection on subcutaneous SK-N-AS and IMRtumors in CB17 SCID mice. Tumors were injected once with either 25 or 50μl PV-10 [8] and monitored daily.

IMR5 tumors were very sensitive to treatment with PV-10 (FIGS. 7A and7B). For control tumors, tumor size increased from 25.6 mm² six dayspost-treatment to 172.9 mm² 23 days post-treatment. By comparison,tumors treated with 25 μl PV-10 increased from 26.6 mm² to 41.2 mm² andtumors treated with 50 μl PV-10 increased from 27.9 mm² to 47.3 mm².Tumor growth was also quantified using a Xenogen IVIS® 200 system thatmeasured bioluminescent signal emitted from tumors, followingintraperitoneal injection of D-Luciferin.

Tumor size decreased following treatment with 25 and 50 μl PV-10 andremained low 17 days post-treatment. Treatment with PV-10 also increasedsurvival, in a dose-dependent manner (FIG. 7C).

Control treated mice had a median survival of 25.5 days, whereas 25 μlPV-10 treated mice survived a median of 41.5 days and 50 μl PV-10treated mice survived a median of 76 days. Additionally, two of the micetreated with 50 μl PV-10 underwent complete tumor regression andremained tumor free for 120 days after treatment.

SK-N-AS tumors also responded to treatment with PV-10 (FIGS. 7D and 7E).For control tumors, tumor size increased from 28.9 mm² six dayspost-treatment to 179.3 mm² 18 days post-treatment. By comparison,tumors treated with 25 μl PV-10 increased from 25.3 mm² to 92.1 mm² andtumors treated with 50 μl PV-10 increased from 29.3 mm² to 57.5 mm².When measured using the Xenogen IVIS® 200 system tumor size decreasedfollowing treatment with 25 and 50 μl PV-10 and remained lower thancontrol treated tumors 15 days post-treatment.

Treatment with PV-10 also increased survival (FIG. 7F). Control treatedmice had a median survival of 29 days, whereas 25 μl PV-10 treated micesurvived a median of 37 days and 50 μl PV-10 treated mice survived amedian of 36 days. Additionally, one mouse treated with 50 μl PV-10underwent complete tumor regression and remained tumor free for 80 daysfollowing treatment.

Discussion

The overall survival rate for children with pediatric solid tumors islower than for children with hematological malignancies [1]. Forchildren with relapsed or metastatic Ewing sarcoma, neuroblastoma,osteosarcoma and rhabdomyosarcoma the overall survival rate is less than30% [1].

Of these cancers, neuroblastoma is the most common and is a leadingcause of death in children aged 1-4 years [2]. Given the poor survivalrates of patients with pediatric solid tumors, and the morbiditiesassociated with the intensive treatment regimens administered tohigh-risk and relapsed patients, there is an urgent need to developnovel therapeutic approaches and early phase clinical trials forpatients with these cancers.

PV-10 is a sterile 10% solution of Rose Bengal (RB,4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein disodium) in 0.9%saline, that induces cell death in a range of adult cancers, but has notpreviously been examined for use on pediatric cancers [7-11]. As PV-10induces cell death in different adult cancers and has been assessed inseveral clinical trials [10, 12, 14, 15, 16], the effects of PV-10 ondifferent pediatric solid tumor cell lines (Ewing sarcoma,neuroblastoma, osteosarcoma and rhabdomyosarcoma) were investigated.

PV-10 decreased cell viability in pediatric solid tumor cell lines in aconcentration-dependent manner. As expected, normal fibroblast celllines and a primary bone marrow sample were less sensitive to PV-10.These data are similar to previously published data on adult cancers [7,9, 11] and indicate that PV-10 could be an effective treatment formultiple pediatric solid tumors.

To characterise the target modulation of PV-10, these studies focused onneuroblastoma, the most common of the extra-cranial solid malignanciesfound in children, and the closely related neuroepithelioma. Byphase-contrast microscopy, PV-10 was found to be cytotoxic toneuroblastoma cells and consistent with IC₅₀ values, SK-N-AS cells wereidentified as most resistant and SK-N-MC most sensitive to PV-10treatment. These findings were verified by time-lapse video microscopythat again showed that SK-N-AS cells were most resistant to treatmentand SK-N-MC were most sensitive.

Additionally, IMR5 cells were found to be more sensitive to treatmentthan SK-N-BE(2) cells at 12 and 24 hours, although by 36 hours few cellsof either type remained. Despite the differences in sensitivity to 100μM PV-10 at early times, this concentration was still cytotoxic to mostSK-N-AS cells at 96 hours and increasing the dose to 200 μM furtherincreased SK-N-AS cell death. These data suggest that PV-10 could be aneffective treatment for all neuroblastomas, although doses may need tobe higher for some sub-types.

The different sensitivities to PV-10 are likely due to the differentgenetic backgrounds of the cell lines as well as the different historiesof the tumors (different patient treatments and primary vs relapsed)from which the cell lines were isolated. Neuroblastoma is a geneticallyheterogeneous disease and the different cell lines were chosen toreflect that heterogeneity.

The most common oncogenic drivers in neuroblastoma are MYCNamplification that is seen in approximately 25% of patients, anaplasticlymphoma kinase (ALK) mutation and amplification that is seen inapproximately 10-15% of patients and mutations in TP53 acquired atrelapse [2]. Using the Catalogue of Somatic Mutations in Cancer (COSMIC)Cell Lines Project [20], it was determined that: SK-N-AS cells hadmutations in NRAS and FLT3; a clone of SK-N-BE(2) had overexpressed ALK,AKT and MYCN along with a homozygous mutation of TP53; IMR5 cellsexhibited over expression of AKT and MYCN with a homozygous mutation ofmTOR; and a clone of SK-N-MC exhibited over expression of MYCN and aheterozygous mutation of TP53. Additionally, SK-N-AS, SK-N-BE(2) andSK-N-MC cell lines were all derived from metastatic tumors withdifferent treatment histories, whereas the IMR5 cell line was derivedfrom a primary tumor.

It has previously been shown that PV-10 acts by disrupting lysosomes,leading to cell death [6]. The disruption of lysosomes specificallyaffects cancer cell survival because cancer cells have an alteredmetabolism and can depend on lysosomes for the recycling of nutrientsand removal of the products of rapid growth and division, such asaggregated proteins and damaged organelles [21]. Furthermore, thedisruption of lysosomes releases cathepsin proteases that can lead toinduction of necrosis or apoptosis [21].

To investigate the effect of PV-10 on lysosomes in neuroblastoma, cellsstained with LysoTracker® Green DND-26, which concentrates andfluoresces in acidic organelles, were observed. Similar to previousresults, lysosomes were found to appear as distinct foci in PBS-treatedcells but were absent in SK-N-BE(2) and IMR5 cells treated with PV-10.Interestingly, in the more resistant SK-N-AS cells, lysosomes were notdisrupted and appeared as distinct foci even after treatment.

Treatment with 100 μM PV-10 induced G1-phase cell cycle arrest in IMR5,but not SK-N-AS cells, and induced apoptosis in a dose- and cellline-dependent manner. It has previously been shown that in adultcancers, PV-10 induces cell death by either apoptosis or necrosis [7, 9,11, 14]. In the study where cells were found to die by necrosis, PV-10induced a G2/M cell cycle arrest prior to cell death [7], suggestingthat PV-10 may have different mechanisms of action in cell lines fromdifferent cancers.

Although treatment with single agent PV-10 has demonstrated efficacy inclinical trials and pre-clinical studies with adult tumors, high-riskneuroblastoma patients are treated with multiple chemotherapies andradiation following relapse [2]. The potential use of PV-10 in combinedtreatment regimens with commonly used chemotherapeutic agents wastherefore investigated.

Following initial screens, a sub-cytotoxic dose of PV-10 (50 μM) wasfound to be synergistic with doxorubicin, etoposide and vincristine inall cell lines studied. Additionally, pre-treatment with PV-10 beforeirradiation improved the efficacy of radiation treatment for bothSK-N-AS and IMR5 cells. These data are consistent with previous datawhere, in a Phase-II clinical trial, pre-treatment of melanoma patientswith PV-10 followed by radiotherapy, induced tumor regression without alarge increase in cytotoxicity [13]. These results indicate that PV-10can be effectively combined with different commonly used treatments, tobenefit high-risk patients with relapsed neuroblastoma.

Having identified that PV-10 was cytotoxic to pediatric solid tumor celllines in vitro, the activity of PV-10 in vivo was examined usingsubcutaneous neuroblastoma xenografts in mice. It was found thatpharmacologically relevant doses [12, 13, 16] of PV-10 induced tumorregression and increased survival in a dose- and tumor-dependent manner.Intralesional injection of 25 and 50 μl PV-10 induced early tumorregression, with 50 μl PV-10 increasing overall survival in mice witheither SK-N-AS or IMR5 tumors. These data are similar to those fromprevious studies using animal models, where intratumoral injection ofPV-10 induced regression of subcutaneous syngeneic colon tumors [7],syngeneic subcutaneous breast tumors and melanoma [14, 8].

In summary, the present studies provide preclinical proof-of-conceptdata on the efficacy of PV-10 in successfully treating pediatric solidtumors (Ewing sarcoma, neuroblastoma, osteosarcoma, andrhabdomyosarcoma). By focusing on neuroblastoma, it is found that PV-10acts by disrupting lysosomes, arresting cells at G1-phase of the cellcycle and inducing apoptosis. Several commonly used treatments have beenidentified with which PV-10 shows synergistic activity. Furthermore, theefficacy of PV-10 treatments in vivo has been validated usingneuroblastoma xenograft mouse studies.

The findings carried out in representative cell lines and in in vivoimmunocompromised mice provide evidence for direct cytotoxic potentialas well as mechanisms by which this agent can induce target modulatoryeffects in cancer cells. Agents that can be combined to generatetreatment synergy have also been identified, providing the framework forthe formulation of early phase clinical trials. This in addition to theexpected immune stimulatory effect described previously, providingsupport for a potential approach where a PV-10 backbone regimen can becombined with agents such as immune check point inhibitors to furtherenhance its activity in patients with relapsed or refractory pediatricsolid tumors.

Materials and Methods

Cell Lines and Tissue Culture

Cell lines (SK-N-AS, SK-N-BE(2), IMR5, LAN1, SK-N-MC, SK-N-SH, SHEP, BJ,BJ hTERT, WI38, WI38 hTERT, Hs68 hTERT, RD, RH30, 143B, HOS, SK-ES andSK-PN-DW) were cultured in Dulbecco's Modified Eagle Medium (DMEM)(Gibco, ON, Canada) supplemented with 5% (v/v) heat inactivated fetalbovine serum (FBS) (Gibco), 100 units/ml penicillin and 100 units/mlstreptomycin (Gibco). Cell cultures were maintained at 37° C. in ahumidified incubator with 5% CO₂. The primary bone marrow sample wasobtained after local Research Ethics Board (REB) approval and writteninformed consent (Ethics ID #17184). Lymphocytes were isolated from thebone marrow sample by density gradient centrifugation using Ficoll-PaquePlus (GE Healthcare Life Sciences, ON, Canada), as described previously[17].

Materials and Reagents

PV-10 (10% solution of Rose Bengal disodium in 0.9% saline) was providedby Provectus Biopharmaceuticals Inc. (Knoxville, Tenn., USA) and storedin the dark at room temperature. Stock solutions of doxorubicin,etoposide, vincristine, cisplatin, pegasparaginase, irinotecan andcytarabine were obtained from the Alberta Children's Hospital Pharmacy(Calgary, AB, Canada) and stored at room temperature in the dark. Forsubsequent studies, the drugs were diluted in DMEM plus supplements tothe appropriate concentrations.

Cytotoxicity Assays

Cells were seeded in 96-well plates (Greiner Bio-One, NC, USA) at 5×10³per well in 100 μl DMEM and cultured for 24 hours. PV-10 alone orphosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄,1.8 mM KH₂PO₄, pH 7.25) (vehicle control) were diluted in DMEM and a 100μl treatment was added to each well. All treatments were run intriplicate at final concentrations ranging from 3.125 to 400 μM.

Plates were cultured for 96 hours. Wells were washed twice with PBS, 200μl fresh DMEM was added to each well and cell viability was evaluatedusing the alamar blue® (Invitrogen, ON, Canada) cytotoxicity assay asper manufacturer's instructions. One-half maximal inhibitoryconcentrations (IC₅₀) were determined using CompuSyn software (ComboSynInc.).

Light Microscopy

Cells were seeded in 6-well plates (Corning Inc., NY, USA) at 2×10⁵ perwell and cultured for 24 hours. Cells were treated with either PBS(vehicle control) or PV-10 and cultured for 96 hours. Phase-contrastimages were captured on a Zeiss Axiovert 200M microscope with a ZeissAxioCam MRm Rev.3 FireWire camera using Zeiss AxioVision Se64 software.Images were processed using Adobe Photoshop (Adobe Creative Cloud 2017).

Time-Lapse Video Microscopy

Cells were seeded in 96-well plates (Greiner Bio-One) at 5×10³ per welland cultured for 24 hours. Cells were treated with either PBS (vehiclecontrol) or PV-10. Three images per well were captured every 30 minutesfor 48 hours using an IncuCyte® Zoom microscope and IncuCyte® Zoomsoftware (Essen BioScience, MI, USA) located in a humidified incubatorwith 5% CO₂ at 37° C. Cell numbers in each well were counted usingImage) software and normalized to cell number at zero hours. At least350 cells were counted per treatment per experiment.

Lysosome Detection and Fluorescence Microscopy

Cells were seeded onto sterile coverslips in 6-well plates (Corning) at2×10⁵/well for not treated cells and 6×10⁵/well for treated cells in6-well plates (Corning) and cultured for 24 hours. Cells were treatedwith either PBS (vehicle control) or PV-10 for 16 hours. Wells werewashed twice with PBS and 2 ml DMEM containing 2.5 μg/ml Hoechst 33342stain (Invitrogen) was added to each well. Cells were incubated at 37°C. for ten minutes and then Lysotracker® Green DND-26 (Invitrogen) wasadded to the media at 500 nM final concentration. Cells were incubatedat 37° C. for 15 minutes, coverslips were mounted onto glass slides attime of imaging and images were captured on a Zeiss Axiovert 200Mmicroscope with a Zeiss AxioCam MRm Rev.3 FireWire camera using ZeissAxioVision Se64 software. Images were processed using Adobe Photoshop(Adobe Creative Cloud 2018).

Flow Cytometry

To analyse cell cycle alterations, cells were seeded in 100 mm dishes(Corning), so that a minimum of 2×10⁶ cells could be collectedpost-treatment. Cells were cultured for 24 hours, treated with eitherPBS (vehicle control) or PV-10 and cultured for either 16 or 24 hours.Cells were collected by trypsinization, washed with PBS, filteredthrough a 40 μm nylon cell strainer (Falcon, Corning, NY, USA), countedusing trypan blue staining using a haemocytometer, re-suspended in 0.9%(w/v) sterile NaCl and fixed in ice-cold 90% (v/v) ethanol. Samples wereincubated at room temperature for 30 minutes then stored at −20° C.

For analysis, samples were centrifuged at 1400 rpm for five minutes at4° C. and washed twice with ice cold PBS. Cells were then incubated at37° C. for 20 minutes in 300 μl labelling buffer: 10 μg/ml DAPI (Sigma,ON, Canada), 200 μg/ml RNase A (Sigma) in 0.1% Triton X-100 in PBS.Samples were run on a BD Bioscience LSR II cytometer using Diva 6.1.3software (BD Bioscience). Results were analyzed using ModFitLT™ 3.3software (Verity Software House).

Preparation of Cellular Extracts

Cells were seeded at 1×10⁶ in 100 mm dishes (Corning) and cultured for24 hours. Cells were then treated with either PBS (vehicle control) orPV-10 and cultured for 24 hours. Medium was collected from cellcultures, cells were washed with PBS and collected followingtrypsinisation. Cells were washed in ice cold PBS and centrifuged at1200 rpm at 4° C. for five minutes. The supernatant was removed, and thepellet was resuspended in radioimmunoprecipitation assay (RIPA) buffer(50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodiumdeoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS)) supplemented with1% (v/v) phosphatase inhibitor (Sigma) and 1% (v/v) protease inhibitor(Sigma). Samples were transferred to 1.5 ml tubes, incubated on ice forten minutes, vortexed and centrifuged at 12,000 rpm for ten minutes.Supernatants were collected as whole cell lysates and either usedimmediately or stored at −20° C.

Western Blotting

Western blotting was carried cut as described previously [17]. Briefly,proteins were transferred to nitrocellulose membrane using a Trans-Blot®Turbo™ Transfer System (BioRad, QC, Canada), transfer was confirmedusing Ponceau S stain (0.1% (w/v) in 5% (v/v) acetic acid) and membraneswere blocked in 5% skim milk in Tris-buffered saline with 0.1% (v/v)Tween®-20 (TBS-T; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v)Tween®-20) at room temperature for two hours. Membranes were thenincubated overnight (about 18 hours) at 4° C. with the following primaryantibodies diluted in 5% (w/v) skim milk in TBS-T: Anti-PARP (1:3000,Cell Signaling, 9542S), anti-caspase 3 (1:500, Cell Signaling, 9662S),anti-caspase 7 (1:1000, Cell signaling, 9492S), anti-caspase 9 (1:1000,Cell Signaling, 9502S) and anti-P-actin (1:5000, Cell Signaling, 8457L).Membranes were washed three times with TBS-T, incubated with anti-rabbitsecondary antibody (1:3000, Cell signaling, 7074S), washed three timeswith TBS-T, incubated with Western Lightning Plus-ECL reagent(Perkin-Elmer, MA, USA) for two minutes and developed using thechemiluminescence setting on a ChemiDoc MP Imaging System (BioRad).

Combination Screens

Cells were cultured as described for cytotoxicity assays. Test drugs(doxorubicin, etoposide, vincristine, cisplatin, pegasparaginase,irinotecan, cytarabine) were prepared at a final concentration of 0.1 μMin media containing either PBS (vehicle control) or PV10 (50 μM final).Treatments were added to cells in triplicate. Plates were cultured,washed and cell viability analyzed by alamar blue®, as described forcytotoxicity assays.

Combination Studies

Cells were cultured as described for cytotoxicity assays. A dilutionseries of three test drugs (doxorubicin, etoposide, vincristine) wereprepared in DMEM containing either PBS (vehicle control) or PV10 (50 μMfinal) and added to cells in triplicate. Plates were cultured, washedand cell viability analyzed by alamar blue®, as described forcytotoxicity assays. Combination indices (CI) for IC₅₀ of test drug incombination with 50 μM PV-10 were calculated using CompuSyn software(ComboSyn Inc.). CI values were scored according to the followingcriteria CI<1 indicated synergistic activity, CI=1 indicated additiveactivity and CI>1 indicated synergistic activity [18].

Radiosensitivity Assays

Cells were seeded at 5×10⁴ in 60 mm dishes (Corning) and incubated for24 hours and treated with either PBS (vehicle control) or 50 μM PV-10and incubated at 37° C. for four hours. Cells were irradiated witheither 0.5, 1 or 2 Gray (Gy) using a Gammacell® 1000 Elite (MDS Nordion,ON, Canada) and cultured for 92 hours. Treatments were run intriplicate. Dishes were washed twice with PBS and cell viability wasanalyzed by alamar blue®, as described for cytotoxicity assays.

In Vivo Xenograft Models

All animal procedures were carried out in accordance with the guidelinesof the Canadian Council on Animal Care and the NIH guidelines on thecare and use of laboratory animals. All protocols were reviewed andapproved by the Animal Care Committee of the University of Calgary(Protocol approval number: AC16-0243).

IMR5-mCherryFluc and SK-N-AS-mCherryFluc cells were used in the animalstudies. These cell lines stably expressed enhanced firefly luciferaseand mCherry on a self-inactivating lentiviral vector encoding theinternal U3 region from murine stem cell virus (mscv), enhanced fireflyluciferase (effLuc), the internal ribosomal entry site (IRES) elementfrom encephalomyocarditis virus (emcv), and mCherry.

Six to eight-week-old female CB17 SCID mice (Charles River Laboratories,QC, Canada) were subcutaneously injected in the right flank with 2.5×10⁶cells (SK-N-ASmCherryFluc or IMR5mCherryFluc) suspended in 0.1 mlMatrigel® Matrix (Fischer Scientific, ON, Canada) (Day zero). Seven daysafter tumor injection, animals with detectable tumor growth of at least5×5 mm were randomised into treatment groups. The groups were treatedwith either 50 μl PBS (vehicle control), 50 μl PV-10 or 25 μl PV-10 byintratumoral (intralesional) injection, according to a previouslyestablished protocol [8]. Animals were monitored daily and tumor areaswere determined with a Vernier caliper. When tumors reached the definedend-point of 15×15 mm, mice were euthanized. Animals that remainedtumor-free were kept for 120 days post-treatment.

Tumor growth was also monitored using the Xenogen IVIS® 200 system(Xenogen Corporation, CA, USA). Mice were imaged to documentbioluminescent signal emitted from tumors, following intraperitonealinjection of D-Luciferin (Gold Biotechnology, MO, USA). Data wereanalyzed by determining total photon flux emission (photons/s) in theregion of interest, as per established methods [19].

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1. A method of treating a pediatric cancerous solid tumor in a mammaliansubject that comprises intralesionally administering an amount of ahalogenated xanthene or a pharmaceutically acceptable salt thereof thatelicits ablation of tumor cells.
 2. The method according to claim 1,wherein said halogenated xanthene molecule is Rose Bengal disodium. 3.The method according to claim 1, wherein said mammal is a human.
 4. Amethod of treating a pediatric cancerous solid tumor in a mammaliansubject that comprises (1) intralesionally administering an amount of ahalogenated xanthene or a pharmaceutically acceptable salt thereof thatelicits ablation of tumor cells and (2) administering a tumor-inhibitingeffective amount of a systemic anti-cancer medication that providessynergistic cytotoxicity with said halogenated xanthene.
 5. The methodaccording to claim 4, wherein said systemic anti-cancer medication isselected from the group consisting of a small molecule, a proteinaceousmolecule, ionizing radiation therapy and checkpoint inhibitorantibodies.
 6. The method according to claim 4, wherein said smallmolecule anti-cancer medication inhibits cellular mitosis.
 7. The methodaccording to claim 4, wherein said proteinaceous anti-cancer medicationinhibits inflammatory chemokine activity.
 8. The method according toclaim 4, wherein said checkpoint inhibitor antibodies inhibit the PD-1receptor, the PD-L1 ligand or the CTLA-4 receptor.
 9. The methodaccording to claim 4, wherein, said systemic anti-cancer medication isprovided by ionizing radiation.
 10. The method according to claim 4,wherein said halogenated xanthene molecule is Rose Bengal disodium. 11.The method according to claim 4, wherein said intralesionaladministration of a halogenated xanthene or a pharmaceuticallyacceptable salt thereof occurs prior to administration of said systemicanti-cancer medication.
 12. The method according to claim 4, whereinsaid intralesional administration of a halogenated xanthene or apharmaceutically acceptable salt thereof occurs after administration ofsaid systemic anti-cancer medication.
 13. The method according to claim4, wherein said intralesional administration of a halogenated xantheneor a pharmaceutically acceptable salt thereof occurs concurrent withadministration of said systemic anti-cancer medication.
 14. The methodaccording to claim 4, wherein said mammal is a human.